The obligate intracellular bacterium Chlamydia trachomatis is a widely disseminated obligate pathogen that infects epithelial surfaces of the conjunctiva and urogenital tract, leading to severe sequela such as blinding trachoma, pelvic inflammatory disease, and infertility. Chlamydia modulates multiple host cellular functions by delivering a large number (>80) of Type III secreted (T3S) effector proteins into the target host cell, whose secretion is regulated by specialized T3S chaperones that stabilize the secretory cargo and enhance their delivery to host cells. We hypothesize that in addition to these canonical functions, Chlamydia T3S chaperones can act as sensors of intracellular stimuli and impart a hierarchy to the secretion of effector proteins. We propose to elucidate the molecular basis and functional role of environmental stimulus-triggered recognition and release of inclusion membrane proteins, a subset of T3S effectors, by the chaperone Mcsc. Preliminary structural and biochemical analysis indicates that pH is an important intracellular signal that modulates the interaction between Mcsc and its effectors. We will test a model wherein an influx of protons due to the proton motor force triggers the rapid release of effectors from Mcsc to facilitate secretion. We will use a combination of mutagenesis, isothermal titration calorimetry, H/D exchange mass spectrometry, NMR, and the newly developed molecular genetic tools to define the binding interfaces of the oligomeric form of Mcsc and its functional implication in Chlamydia. We also propose to elucidate the molecular function of Slc1, the most abundant T3S chaperone in Chlamydia. We will examine if the temporal regulation in the secretion of effectors early in infection is determined by the differential binding affinities of Slc1 towards its effecto cargo proteins. By performing domain swapping experiments and point mutagenesis in key residues determining binding affinities, we will determine the general rules that govern chaperone-mediated temporal control of secretion. In addition, we will explore the significance of Slc1 interactions with the T3S basal component CdsD to assess its role in establishing a hierarchy in the translocation of effectors early during infection. Efficient interactions between T3S chaperones and secretion cargoes are important for stabilizing effector proteins and for regulating their orderly secretion, but these interactions also present an energy barrier that impedes rapid cargo release. How T3S chaperones regulate this process in response to environmental stimuli is not known. Our proposed structure-function analysis of Mcsc and Slc1 will unveil exciting new features of T3S chaperones and will shed fresh insights on how these conserved proteins regulate a key step in bacterial pathogenesis.